The present invention relates to methods for predicting and following illnesses. More particularly, the present invention relates to the diagnosis of preeclampsia and other diseases.
Vascular disease is often related to the composition of blood flowing therethrough. In particular, high concentrations of very low density lipoproteins (VLDL) in blood have a deleterious effect on vascular integrity. Very low density lipoproteins in blood tend to break down the inner vascular walls causing vascular diseases including preeclampsia, atherosclerosis, stroke, peripheral vascular disease, diabetic vascular disease, and such.
Methods providing earlier detection of vascular diseases, and methods for diagnosing a patient""s proclivity toward developing a vascular disease at a later point in his life are desirable so that such disease may be better controlled, or even avoided. The early detection of preeclampsia is particularly important.
Preeclampsia is a toxic vascular disease of particular interest. Preeclampsia develops in late pregnancy and is characterized by a sudden rise in blood pressure, excessive increase in weight, generalized edema, albuminuria, severe headaches, and visual disturbances. The blood vessels in a pregnant woman""s uterus supplying blood to her placenta and fetus become restricted during preeclampsia, thereby delivering reduced amounts of blood and oxygen to the fetus. Preeclampsia is linked to poor fetal growth and, in its most severe form, can be fatal to both the fetus and the mother.
Human blood""s natural defense against the destructive effect of VLDL on Endothelial cells and Leukocytes has been quantified by an index or factor known as the xe2x80x9ctoxicity preventing activityxe2x80x9d or xe2x80x9cTxPAxe2x80x9d of the blood. (Arbogast, B. W., and Dreher, N. J. Coronary Disease Prediction Using a New Atherogenic Index. Atherosclerosis, Vol. 73 (1988) 259-267). (Chi, D. S., et al. Decreased Lymphocyte Response in Streptozotocin Induced Diabetic Rats: A Function of Very Low Density Lipoproteins. Diabetes 31 (1982) 1098-1104). U.S. Pat. No. 4,699,878 discloses that the TxPA of a blood sample can be estimated by comparison of the growth of a culture of cells treated with a toxic quantity of VLDL and varying amounts of the blood sample to the xe2x80x9czeroxe2x80x9d growth of a reference culture of cells which was treated with the same toxic quantity of VLDL and no blood.
The ratio of VLDL to TxPA determines the cytotoxicity of the blood in in vitro cell cultures. The ratio of in vitro VLDL to TxPA has also been effective in determining in vivo vascular injury. The presence of or future development of preeclampsia can be predicted with a 90% accuracy using the ratio of VLDL to TxPA. (Arbogast, B. W., Leeper, S. C., Merrick R. D., Olive, K. E. and Taylor, R. N Plasma Factors that Determine Endothelial Cell Lipid Toxicity in vitro Correctly Identify Women with Preeclampsia in Early and Late Pregnancy. Hypertension in Pregnancy Vol. 15 (1996) 263-279). Similar accuracy has been achieved with atherosclerosis. (Arbogast, B. W., Gill, L. R. and Schwertner, H. A. A New Protective Factor in Coronary Artery Disease: Very-Low-Density Lipoprotein Toxicity-Preventing Activity. Atherosclerosis Vol. 57 (1985) 75-86). (Arbogast, B. W. and Dreher, N. J. Coronary Disease Prediction Using a New Atherogenic Index. Atherosclerosis Vol. 66 (1987) 55-62). The drawbacks of this cell culture method are that it is a relatively expensive assay and that it requires cell growth time. Also, the level of uncertainty is about 10%, undesirably high for a medical assay.
Blood plasma contains components including albumin, non-esterified fatty acids (NEFA), and triglycerides which are carried in varying amounts on very low density lipoproteins (VLDL), low density lipoproteins (LDL), and high density lipoproteins (HDL). Human blood albumin exists as two species that may be separated by their electrophoretic migration to isoelectric points of pH 4.8 and pH 5.6. (Basu, S. P., Rao, S. N. and Hartsuck, J. A. Influence of Fatty Acid and Time of Focusing on the Isoelectric Focusing of Human Plasma Albumin. Biochim Biophys Acta, Vol. 533 (1978) 66). It has been found that the toxicity preventing activity of human blood is mainly provided by pI 5.6 albumin. Arbogast disclosed that the pI 5.6 albumin species provides the protective effect against VLDL damage to vasculature endothelial cells and leukocytes. (Arbogast, B. W. Purification and Identification of Very Low Density Lipoprotein Toxicity Preventing Activity. Atherosclerosis Vol. 7 (1988) 259-267 and Chi, D. S., Berry, D. L., Dillon, K. A. and Arbogast, B. W.: Decreased Lymphocyte Response in Streptozotocin Induced Diabetic Rats: A Function of Very Low Density Lipoproteins. Diabetes 31: 1098-1 104, 1982). Accordingly, the determination of the pI 5.6 albumin concentration in blood would be greatly beneficial in diagnosing vascular and leukocyte associated diseases.
The concentration of pI 5.6 albumin is not determinable from the total albumin concentration, due to the fact that the pI 5.6 and pI 4.8 albumin species exist in human plasma in unpredictable ratios. The TxPA of a sample of plasma can be determined by separating the pI 4.8 albumin from the pI 5.6 albumin via liquid column isoelectric focusing and determining the concentration of the pI 5.6 albumin fraction via absorbance spectrometry. The concentration of pI 5.6 albumin in plasma is then compared to a standard concentration known to indicate delineation between patients having been diagnosed with arterial disease and those not diagnosed with arterial disease. (Arbogast, B. W., Leeper, S. C., Merrick, R. D., Olive, K. E. and Taylor, R. N.: Plasma Factors that Determine Endothelial Cell Lipid Toxicity in vitro Correctly Identify Women with Preeclampsia in Early and Late Pregnancy. Hypertension in Pregnancy 15:263-279, 1996). The electrophoretic method disclosed by Arbogast is quite cumbersome and expensive for clinical operation. The degree of uncertainty of the electrophoresis method is about 10%.
In light of the above, it would be desirable to have a simpler process for diagnosing the presence of or the proclivity toward developing albumin-inhibited VLDL-sensitive diseases, including vascular and non-vascular diseases and conditions. A process not requiring cell culture growth or an isoelectric focusing separation would be more useful would be further desirable for such new diagnostic process to be more accurate than previous processes.
The present invention is a process for determining the toxicity preventing ability of plasma against a disease having a correlation to a reduction in the concentration of pI 5.6 albumin in the plasma. The present process comprises the steps of:
(a) providing a plasma sample containing free albumin, free non-esterified fatty acids, triglycerides, very low density lipoproteins, low density lipoproteins, and high density lipoproteins;
(b) determining the concentration of the free albumin;
(c) determining the concentration of the free non-esterified fatty acids; and
(d) calculating a value indicative of the toxicity preventing ability of the plasma by comparing the concentration of the free albumin to the concentration of the free non-esterified fatty acids. The preferred indicator value is a xe2x80x9cTxPA-S ratioxe2x80x9d, calculated by dividing the concentration of the free albumin by the concentration of the free non-esterified fatty acids.
The present invention further includes an assay kit useful for conducting the present process. The assay kit comprises the following:
(a) a lipid-precipitating reagent;
(b) a reagent that displays a color upon binding with albumin; and
(c) a reagent that displays a color upon binding with non-esterified fatty acids.